Methods
RPE/BrM-only flat-mounts from 11 belts of normal chorioretinal human tissue (5 donors < 50 yrs, 6 > 80 yrs; 8 females, 3 males) were prepared by removing the retina and choroid under photographic control for maintaining foveal position. Spectral microscopy and hyperspectral AF imaging were performed at 2 excitation bands, 436-460nm and 480-510nm, with emissions captured using the Nuance FX camera (Caliper Life Sciences, US) between 420-720nm in 10nm intervals at 3 locations: fovea, parafovea (2-4mm superior to fovea, at the rod peak) and periphery (8-10mm superior, at the highest rod:cone ratio), giving 66 hyperspectral data sets, consisting of photon counts per second recorded at each spatial pixel in the 40X field and wavelength.

Results
Gaussian mixture modeling and mathematical factorization of the hypercubes were applied to extract four RPE candidate spectra for lipofuscin at each location for each donor (see abstract by Johri et al for details). The four peaks were seen at average wavelengths of 566±6nm, 604±27nm, 645±8nm, and 701±8nm at the 436-460nm excitation (Fig. 1) and 558±8nm, 606±5nm, 646±8nm and 694±13nm at the 480-510nm excitation across all donors. The peak near 600nm (A2E-like) was generally the smallest peak amongst the four. The emission maxima varied for donors across age and locations, but all spectra were present in all but 6/108 data sets. There were no consistent regional or age trends in peak intensities.

Conclusions
Hyperspectral AF imaging analysis of the RPE ex vivo consistently reports the presence of at least 4 abundant fluorophors with well-defined emission maxima across all studied ages, retinal locations, and excitation wavelengths. Determining the actual abundant source molecules that produce these signals will be important in understanding RPE physiology.